| Soybean is main oil crop in China and also an important source of plant protein for human beings.However,soybean yield per unit area in China is less than 60% of that in the United States of America,and China is a major soybean importer in the world.The key to revitalize China’s soybean industry is to increase its yield per unit area.Although seed number per pod in soybean is an important component of soybean yield,it keeps stably across wild,landrace and bred soybeans,being only 1～4 seeds,which is much significantly lower than that in cowpea.To increase soybean yield,therefore,it is important to dissect the molecular mechanisms for the difference of seed number per pod between soybean and cowpea using bioinformatics approaches.Seeds are derived from the fertilization of ovule,as we know,the formation of ovule in Arabidopsis thaliana is regulated by auxin and cytokinin,and the two hormone pathways are closely linked by PIN1 protein.PIN1,as an efflux transporter of auxin,controls auxin transport and plays a crucial role in the formation of the auxin maximum zone at the apex of the ovule primordia.The positive regulation of ovule formation by cytokinin is achieved by activating PIN1 expression,but cytokinin is degraded by the CKX protein family.To dissect the molecular mechanisms for the difference of seed number per pod between soybean and cowpea,thus,in this study we identified the gene families of PIN1 and CKX in soybean and cowpea,and carried out bioinformatics analysis for the two gene families.The results would provide new ideas for improving soybean yield.The main results are as follows:1.Among five soybean Gm PIN1 genes and three cowpea Vu PIN1 genes identified by homologous alignment of gene PIN1 in Arabidopsis,Gm PIN1 a and Gm PIN1 b in soybean and Vu PIN1 b in cowpea were found by evolutionary,motif and collinear analyses to be key PIN1 genes in the auxin maxima zone of ovule primordia.Among the three key PIN1 genes,the relative expression levels of Gm PIN1 a and Gm PIN1 b in soybean flowers were much lower than that of Vu PIN1 b in cowpea.In the STRING database some Arabidopsis proteins,with the evidence of molecular biology,were found to be interacted with the PIN1 protein,and the relative expression levels of the soybean and cowpea homologous genes of these interacting proteins were compared.As a result,the low expression levels of PID and D6 PK kinases gene in soybean Gm PIN1 and the high expression level of regulatory subunit RCN1 gene by phosphatase caused the lower phosphorylation level of Gm PIN1 in soybean than that of Vu PIN1 in cowpea.The high expression levels of UNH gene,which promotes the degradation of Gm PIN1 in marginal cells,and MPK6 gene,which affects its plasma membrane localization,resulted in the lower plasma membrane localization level of Gm PIN1 in soybean than that of Vu PIN1 in cowpea.We deduced that the significant differences for relative expression,phosphorylation,and plasma membrane localization levels of the PIN1 proteins(or genes)in soybean and cowpea affects the formation rate of auxin maxima zone in ovule primordia in soybean and cowpea,and the number of ovules per pistil and seed number per pod.2.Among seventeen CKX soybean genes and ten CKX cowpea genes identified by homologous alignment of gene CKX in Arabidopsis,their properties were revealed by evolutionary,gene structure,motif,and cis element analyses,and physical and chemical property prediction.Among the twenty-seven genes,CKX5 had incomplete domain and no expression level in soybean,and the relative expression levels of CKX3,CKX6 and CKX7 in soybean were much higher than those in cowpea.By comparing the relative expression levels of soybean and cowpea genes,which are homologous to ROCK1 that positively regulates CKX protein activity in Arabidopsis thaliana,and predicting the mi RNAs that regulate the CKX gene family in soybean and cowpea,higher CKX protein activity owing to high expression level of ROCK1 in soybean than that in cowpea,and higher expression levels of CKX gene family owing to a few mi RNAs in soybean resulted in more cytokinin degradation,which reduces the relative expression level of PIN1 gene in soybean,decreasing seed number per pod.Based on the evolutionary analysis of Gm CKX gene family,the expansion of Gm CKX gene family was mainly derived from whole-genome duplication events of soybean in 8 ～13 MYA and 59 MYA,the tandem duplication event of Gm CKX3 occurred in 59 MYA,and the single fragment duplication of Gm CKX1 occurred in 59 MYA and the duplication evolved into Gm CKX6.Soybean genes homologous to CKX3 in Arabidopsis named Gm CKX3 a,while new genes derived from their tandem duplications named Gm CKX3 b.In the homologous alignment of CKX gene family in cowpea,kidney bean,chickpea and Medicago truncatula,almost all the legume crops experienced tandem duplication of CKX3,and the tandem duplication gene Medtr4G126160 of Medicago truncatula gene Medtr4G126150 homologous to CKX3 in Arabidopsis thaliana is a CKX3 b gene,which affects the root development of Medicago truncatula.Based on the expression patterns of different tissues for CKX gene family in soybean,cowpea and kidney bean and results in previous studies,a key gene,CKX3 a,affecting the number of ovules was highly expressed in flowers;the CKX3 b genes weren’t expressed in flowers;some genes were highly expressed in soybean and Medicago truncatula roots and might affect root development,such as Medtr4G126160;some genes weren’t expressed in almost all the tissues,such as in cowpea and kidney bean.The CKX6 and CKX7 genes were highly expressed in flowers,which might have the same function as the CKX3 a gene.Based on the results that the ckx3 ckx5 mutants in Arabidopsis thaliana and Brassica napus can increase the number of ovules per pistile,the number of pods and yield,along with incomplete gene domain and no expression of CKX5 and the functional differentiation of CKX3 b in soybean,we deduce that the ckx3 a mutant or the ckx3 a ckx6ckx7 mutants may increase the seed number per pod and yield. |
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